Fluorometer for flow injection analysis with application to oxidase enzyme dependent reactions

Abstract

A slmple effective flow fluorometer has been assembled and operated In conjunctlon wlth a flow Injection analysls (FIA) system. A submlcrollter caplllary sheath flow cell Is used for fluorescence detection wlth a laser source. The Instrument has been tested for stablllty, preclslon, accuracy, sensitivity, dynamic range, and speed. Rhodamlne 6G was used as a standard fluorophore. Appllcatlon was also made to the cllnlcal analysis of oxidase enzyme dependent reactlons by reactlng H202 with leucodlacetyldlchlorofluoresceln to produce fluorescent dichlorofluoresceln. Thls produced dye is excited at 488 nm wlth the argon laser. Concentratlons of lo-'' M hydrogen peroxide could be detected when Injecting 25 pL allquots.

Rejection of capillary wall background light in an on-column confocal fluorescence detector

“…The present detection sensitivity is just slightly lower than that with the sheath-flow fluorescent detector used in previous studies, 21,22 but this on-column confocal detector is easier to use and more robust than a sheath-flow system. In the sheath-flow cuvette, a buffer stream is used to ensheath the analytes eluted from the capillary.…”

Analytical Utilization of Phenylfluorone as a Hydrogen Donor Substrate for Peroxidase

“…A great many procedures have been proposed to determine hydrogen peroxide. Examples include electrochemical methods (3,4), spectrophotometric methods (5,6), fluorescence techniques (7,8), and chemiluminescence methods (9,10). Nowadays, the most widely used methods for the determination of hydrogen peroxide in clinical chemistry are the spectrophotometric procedures by reaction with a chromogenic hydrogen donor substrate in the presence of peroxidase (2).…”

Application ofo-Hydroxyphenylfluorone as a Fluorogenic Indicator to the Determination of Hydrogen Peroxide and Oxidase Substrates Based on the Catalytic Effect of Hemin

“…These include chemiluminescence (2,3), absorption photometric methods (4)(5)(6), electrochemical methods (7,8), and fluorescence techniques (9,10). At present, the most widely used fluorometric methods for the determination of H 2 O 2 are based on the formation of a fluorescent oxidation product (11,12) and rely largely on p-hydroxyphenylacetic acid (HPA) and homovanillic acid (HVA) as the fluorescent substrates in the presence of peroxidase (13)(14)(15)(16). However, peroxidase is expensive and its solution is not stable.…”